Currently available antidepressants work well for only a subset of individuals with depression and other stress-related disorders. Therefore, identifying new targets for treating depression is an urgent health imperative. The Lateral Habenula (LHb) is a small, bilateral structure that appears to play a critical role as a relay node connecting several key brain regions that are involved in mood control such as the dorsal raphe nucleus (DRN), ventral tegmental area (VTA), and the rostromedial tegmental nucleus (RMTG). The detailed neurochemistry of the LHb is poorly understood. We know that LHb plays a critical role in mediating negative or aversive emotional states relating to mood, reward, and motivation. Pilot studies in humans and animal models suggest that the LHb may be an important target for treating refractory depression. For example, we found that the expression and activation of hM4Di, a Gi-coupled inhibitory DREADD, in LHb produced an antidepressant effect in the forced swim test model of antidepressant action. The primary technical strategy for this project will be the use of intersectional transgene expression in rats using two types of viral vectors: AAV-DIO-transgene vectors (injected into LHb) and CAV2-Cre vectors (injected into one of the projection target regions-DRN, VTA or RMTG). Together these vectors produce transgene expression only in neurons of the desired pathway. This strategy will be used to manipulate the activity of these discrete pathways (with DREADDs) as well as to interrogate mRNA translation in each (with RiboTag). Aim 1 will test which of these LHb pathways is responsible for modulating immobility in the forced swim test. We predict that modulating the LHb to DRN pathway will alter immobility, and that activating the Gi-coupled DREADD with the ligand clozapine-N-oxide will reduce immobility whereas activating the Gs-coupled DREADD will exacerbate immobility. Aim 2 will assess the contribution of these pathways to different domains of stress-related emotional states such as anhedonia (saccharin preference), anxiety (open field), and emotional learning (conditioned place aversion) using DREADDs that activate or inhibit these pathways selectively. Aim 3 will assess how LHb pathways alter the emergence of behavioral vulnerability or resilience in response to repeated social defeat stress. Aim 4 will use RiboTag, an epitope-tagged ribosomal protein, to selectively immunopurify the polyribosomes from the transduced pathways followed by RNAseq and validation of significantly different mRNAs. This will allow us to evaluate the gene expression phenotypes of neurons in each pathway, and to investigate how LHb neurons respond to stress exposure as well as antidepressant treatment. It will be a powerful method for identifying regulatory networks and potential nodes of control that can promote resilience to stress. Together, these experiments will provide a great deal of new information about the functional organization of LHb, its role in animal models of stress, and may help to identify new molecular targets in LHb that can leveraged in the treatment of stress disorders.